Summary. HIV rebound occurs in most HIV infected individuals within weeks of antiretroviral therapy (ART) interruption. Lymphoid tissues (LT, composed of lymph nodes, tonsil, adenoid, splenic white pulp, lymphoid aggregates in peripheral tissues, etc.) are a major site for maintenance and subsequent recrudescence of the long-term HIV reservoir. While our current knowledge continues to be refined, latently infected follicular helper (Tfh) and central memory (Tcm) CD4+ T cells within LT make up the majority of the reservoir in ART treated subjects. Importantly, most T cells are not stationary, and can leave and re-enter LT or other peripheral sites. This process of continual movement of lymphocytes between blood, peripheral tissues, and LT, termed lymphocyte recirculation, is a central process of the immune system. Early studies of lymphocyte recirculation found that the average time a lymphocyte spends in blood is only ~ 60 minutes, and that enough lymphocytes enter the blood from LT to replace all those in blood 11x/day. The impact this rapid and massive migration of lymphocytes has for HIV immunopathogenesis, eradication, and cure remains unstudied. Here we will test whether (1) lymphocyte recirculation allows for the seeding and redistribution of infected CD4+ T cells between sanctuary sites in LT and other peripheral reservoir sites during chronic infection or after ATI, and (2) lymphocyte recirculation mechanisms reduce the potential interaction of CD8+ T cells with infected CD4+ T cells within LT. We will inhibit lymphocyte recirculation in ART-treated SIV-infected rhesus macaques (RM) using the cell migration inhibitor FTY720 [fingolimod, a sphingosine-1 phosphate receptor (S1PR) agonist]. FTY720 inhibits the egress of T and B cells from tissues- especially LT. We hypothesize that FTY720 treatment during ATI in SIV-infected RM will prevent reactivated HIV infected CD4+ T cells from leaving LT and retain newly activated SIV-specific CD8+ T cells in LT. Through the use of FTY720, we will therefore be able to, in Aim 1, define the impact of inhibiting lymphocyte recirculation on viral dynamics following ATI in early and late treated SIV infection. Using barcoded SIVmac239, we will be able to precisely characterize viral rebound kinetics, reservoir changes, reactivation rate, reservoir diversification and synchronization between different tissue, and re-seeding of the reservoir following treatment interruption when only cell-free, rather than cell-associated, virus could mediate these effects. In Aim 2 we will determine whether CD8+ T cells can limit viral reactivation and rebound in LT following ATI in early and/or late treated SIV infection. FTY720 will cause CD8+ T cells to remain in the LT following ART interruption. We can therefore determine whether CD8+ T cells in LT functionally gain the ability to control or eliminate reactivating SIV- infected CD4+ T cells after therapy interruption. Together these studies will provide new insight into the source of viral rebound and potential strategies to control viral rebound.